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Huub Op Den J M Camp - One of the best experts on this subject based on the ideXlab platform.
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high level functional expression of a fungal xylose isomerase the key to efficient ethanolic fermentation of xylose by saccharomyces cerevisiae
Fems Yeast Research, 2003Co-Authors: Harry Ramanoedj Harhangi, Ann Kristin Stave, Mike S M Jetten, Wim T A M De Laat, Jan Den J J Ridder, Huub Op Den J M CampAbstract:Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.
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genomic dna analysis of genes encoding hemi cellulolytic enzymes of the anaerobic fungus Piromyces sp e2
Gene, 2003Co-Authors: Harry Ramanoedj Harhangi, Peter J. M. Steenbakkers, Anna Akhmanova, Mike S M Jetten, Chris Van Der Drift, Huub Op Den J M CampAbstract:Abstract Anaerobic fungi contain more than one copy of genes encoding (hemi-)cellulases in their genome. The arrangement of these genes on the chromosomes was not known. A genomic DNA (gDNA) library of Piromyces sp. E2 was screened with different probes specific for (hemi-)cellulolytic enzymes. This screening resulted in three gDNA clones with genes encoding glycoside hydrolase enzymes of families 1 (β-glucosidase), 6 (exoglucanase) and 26 (mannanase). Each clone contained two or more genes of the same family. Comparison of the gene copies on a clone revealed that they were highly homologous, and in addition, 54–75% of the substitutions was synonymous. One of the mannanase genes contained an intron. PCR with selected primers resulted in a gDNA clone with a new representative (cel9B) of glycoside hydrolase family 9 (endoglucanase). Comparison with cel9A revealed that cel9B had 67% homology on the nucleotide level. Furthermore, three introns were present. All results of this paper taken together provided evidence for duplications of (hemi-)cellulolytic genes, which resulted in clusters of almost identical genes arranged head-to-tail on the genome. In contrast to other eukaryotes, this phenomenon appears frequently in anaerobic fungi.
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beta glucosidase in cellulosome of the anaerobic fungus Piromyces sp strain e2 is a family 3 glycoside hydrolase
Biochemical Journal, 2003Co-Authors: Peter J. M. Steenbakkers, Jan T. Keltjens, Harry Ramanoedj Harhangi, G D Vogels, Chris Van Der Drift, Mirjam W Bosscher, Marlous M C Van Der Hooft, Huub Op Den J M CampAbstract:The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a beta-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular beta-glucosidase activity. To identify the component in the fungal cellulosome responsible for the beta-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737 bp immunoclone was isolated from a cDNA library. The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A. The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components. The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified. Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for beta-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent K (m) and K (i) values of heterologous Cel3A and the fungal cellulosome for p -nitrophenyl-beta-D-glucopyranoside and D-glucono-1,5-delta-lactone respectively indicated that cel3A encodes the beta-glucosidase activity of the Piromyces sp. strain E2 cellulosome.
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noncatalytic docking domains of cellulosomes of anaerobic fungi
Journal of Bacteriology, 2001Co-Authors: Peter J. M. Steenbakkers, Xinliang Li, Eduardo Ximenes, Jorik G Arts, Huizhong Chen, Lars G Ljungdahl, Huub Op Den J M CampAbstract:A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungus Piromyces sp. strain E2 and the polycentric fungus Orpinomyces sp. strain PC-2. Through PCR using cDNA from Orpinomyces sp. and genomic DNA from Piromyces sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an Orpinomyces sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus Orpinomyces sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.
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a hydrogenosome with pyruvate formate lyase anaerobic chytrid fungi use an alternative route for pyruvate catabolism
Molecular Microbiology, 1999Co-Authors: Anna Akhmanova, Ken M Hosea, Frank Voncken, Jan T. Keltjens, Godfried D. Vogels, Harry Ramanoedj Harhangi, Huub Op Den J M Camp, Johannes H P HacksteinAbstract:The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.
Harry Ramanoedj Harhangi - One of the best experts on this subject based on the ideXlab platform.
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high level functional expression of a fungal xylose isomerase the key to efficient ethanolic fermentation of xylose by saccharomyces cerevisiae
Fems Yeast Research, 2003Co-Authors: Harry Ramanoedj Harhangi, Ann Kristin Stave, Mike S M Jetten, Wim T A M De Laat, Jan Den J J Ridder, Huub Op Den J M CampAbstract:Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.
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genomic dna analysis of genes encoding hemi cellulolytic enzymes of the anaerobic fungus Piromyces sp e2
Gene, 2003Co-Authors: Harry Ramanoedj Harhangi, Peter J. M. Steenbakkers, Anna Akhmanova, Mike S M Jetten, Chris Van Der Drift, Huub Op Den J M CampAbstract:Abstract Anaerobic fungi contain more than one copy of genes encoding (hemi-)cellulases in their genome. The arrangement of these genes on the chromosomes was not known. A genomic DNA (gDNA) library of Piromyces sp. E2 was screened with different probes specific for (hemi-)cellulolytic enzymes. This screening resulted in three gDNA clones with genes encoding glycoside hydrolase enzymes of families 1 (β-glucosidase), 6 (exoglucanase) and 26 (mannanase). Each clone contained two or more genes of the same family. Comparison of the gene copies on a clone revealed that they were highly homologous, and in addition, 54–75% of the substitutions was synonymous. One of the mannanase genes contained an intron. PCR with selected primers resulted in a gDNA clone with a new representative (cel9B) of glycoside hydrolase family 9 (endoglucanase). Comparison with cel9A revealed that cel9B had 67% homology on the nucleotide level. Furthermore, three introns were present. All results of this paper taken together provided evidence for duplications of (hemi-)cellulolytic genes, which resulted in clusters of almost identical genes arranged head-to-tail on the genome. In contrast to other eukaryotes, this phenomenon appears frequently in anaerobic fungi.
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cel6a a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp e2 and Piromyces equi
Biochimica et Biophysica Acta, 2003Co-Authors: Harry Ramanoedj Harhangi, Alex Freelove, Peter J. M. Steenbakkers, Anna Akhmanova, Mike S M Jetten, Wimal Ubhayasekera, Maarten Van Dinther, Chris Van Der Drift, Sherry L Mowbray, Harry J GilbertAbstract:Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.
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xylose metabolism in the anaerobic fungus Piromyces sp strain e2 follows the bacterial pathway
Archives of Microbiology, 2003Co-Authors: Harry Ramanoedj Harhangi, Anna Akhmanova, Roul Emmens, Chris Van Der Drif, Wim T A M De Laa, Johannes P Van Dijke, Mike S M Jette, Jack T Pronk, Huub Op De J M CampAbstract:The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min−1mg protein-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.
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beta glucosidase in cellulosome of the anaerobic fungus Piromyces sp strain e2 is a family 3 glycoside hydrolase
Biochemical Journal, 2003Co-Authors: Peter J. M. Steenbakkers, Jan T. Keltjens, Harry Ramanoedj Harhangi, G D Vogels, Chris Van Der Drift, Mirjam W Bosscher, Marlous M C Van Der Hooft, Huub Op Den J M CampAbstract:The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a beta-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular beta-glucosidase activity. To identify the component in the fungal cellulosome responsible for the beta-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737 bp immunoclone was isolated from a cDNA library. The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A. The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components. The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified. Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for beta-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent K (m) and K (i) values of heterologous Cel3A and the fungal cellulosome for p -nitrophenyl-beta-D-glucopyranoside and D-glucono-1,5-delta-lactone respectively indicated that cel3A encodes the beta-glucosidase activity of the Piromyces sp. strain E2 cellulosome.
Anna Akhmanova - One of the best experts on this subject based on the ideXlab platform.
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the anaerobic chytridiomycete fungus Piromyces sp e2 produces ethanol via pyruvate formate lyase and an alcohol dehydrogenase e
Molecular Microbiology, 2004Co-Authors: Brigitte Boxma, Frank Voncken, Jaap J Van Hellemond, Anna Akhmanova, Martijn A Huynen, Sander Jannink, Theo A Van Alen, Susanne W H Van Weelden, Aloysius G M TielensAbstract:Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.
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genomic dna analysis of genes encoding hemi cellulolytic enzymes of the anaerobic fungus Piromyces sp e2
Gene, 2003Co-Authors: Harry Ramanoedj Harhangi, Peter J. M. Steenbakkers, Anna Akhmanova, Mike S M Jetten, Chris Van Der Drift, Huub Op Den J M CampAbstract:Abstract Anaerobic fungi contain more than one copy of genes encoding (hemi-)cellulases in their genome. The arrangement of these genes on the chromosomes was not known. A genomic DNA (gDNA) library of Piromyces sp. E2 was screened with different probes specific for (hemi-)cellulolytic enzymes. This screening resulted in three gDNA clones with genes encoding glycoside hydrolase enzymes of families 1 (β-glucosidase), 6 (exoglucanase) and 26 (mannanase). Each clone contained two or more genes of the same family. Comparison of the gene copies on a clone revealed that they were highly homologous, and in addition, 54–75% of the substitutions was synonymous. One of the mannanase genes contained an intron. PCR with selected primers resulted in a gDNA clone with a new representative (cel9B) of glycoside hydrolase family 9 (endoglucanase). Comparison with cel9A revealed that cel9B had 67% homology on the nucleotide level. Furthermore, three introns were present. All results of this paper taken together provided evidence for duplications of (hemi-)cellulolytic genes, which resulted in clusters of almost identical genes arranged head-to-tail on the genome. In contrast to other eukaryotes, this phenomenon appears frequently in anaerobic fungi.
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cel6a a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp e2 and Piromyces equi
Biochimica et Biophysica Acta, 2003Co-Authors: Harry Ramanoedj Harhangi, Alex Freelove, Peter J. M. Steenbakkers, Anna Akhmanova, Mike S M Jetten, Wimal Ubhayasekera, Maarten Van Dinther, Chris Van Der Drift, Sherry L Mowbray, Harry J GilbertAbstract:Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.
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xylose metabolism in the anaerobic fungus Piromyces sp strain e2 follows the bacterial pathway
Archives of Microbiology, 2003Co-Authors: Harry Ramanoedj Harhangi, Anna Akhmanova, Roul Emmens, Chris Van Der Drif, Wim T A M De Laa, Johannes P Van Dijke, Mike S M Jette, Jack T Pronk, Huub Op De J M CampAbstract:The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min−1mg protein-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.
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a hydrogenosome with pyruvate formate lyase anaerobic chytrid fungi use an alternative route for pyruvate catabolism
Molecular Microbiology, 1999Co-Authors: Anna Akhmanova, Ken M Hosea, Frank Voncken, Jan T. Keltjens, Godfried D. Vogels, Harry Ramanoedj Harhangi, Huub Op Den J M Camp, Johannes H P HacksteinAbstract:The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.
Harry J Gilbert - One of the best experts on this subject based on the ideXlab platform.
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Newcastle upon Tyne, Newcastle upon Tyne
2014Co-Authors: Ruth Y. Eberhardt, Harry J Gilbert, Geoffrey P. HazlewoodAbstract:Primary sequence and enzymic properties of two modular endoglucanases, Cel5A and Cel45A, from the anaerobic fungus Piromyces equ
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characterization of a double dockerin from the cellulosome of the anaerobic fungus Piromyces equi
Journal of Molecular Biology, 2007Co-Authors: Tibor Nagy, Lee D. Higgins, Chris Walters, Richard B. Tunnicliffe, Harry J Gilbert, Michael P WilliamsonAbstract:The assembly into supramolecular complexes of proteins having complementary activities is central to cellular function. One such complex of considerable biological and industrial significance is the plant cell wall-degrading apparatus of anaerobic microorganisms, termed the cellulosome. A central feature of bacterial cellulosomes is a large non-catalytic protein, the scaffoldin, which contains multiple cohesin domains. An array of digestive enzymes is incorporated into the cellulosome through the interaction of the dockerin domains, present in the catalytic subunits, with the cohesin domains that are present in the scaffoldin. By contrast, in anaerobic fungi, such as Piromyces equi, the dockerins of cellulosomal enzymes are often present in tandem copies; however, the identity of the cognate cohesin domains in these organisms is unclear, hindering further biotechnological development of the fungal cellulosome. Here, we characterise the solution structure and function of a double-dockerin construct from the P. equi endoglucanase Cel45A. We show that the two domains are connected by a flexible linker that is short enough to keep the binding sites of the two domains on adjacent surfaces, and allows the double-dockerin construct to bind more tightly to cellulosomes than a single domain and with greater coverage. The double dockerin binds to the GH3 beta-glucosidase component of the fungal cellulosome, which is thereby identified as a potential scaffoldin.
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cel6a a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp e2 and Piromyces equi
Biochimica et Biophysica Acta, 2003Co-Authors: Harry Ramanoedj Harhangi, Alex Freelove, Peter J. M. Steenbakkers, Anna Akhmanova, Mike S M Jetten, Wimal Ubhayasekera, Maarten Van Dinther, Chris Van Der Drift, Sherry L Mowbray, Harry J GilbertAbstract:Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.
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the major component of the cellulosomes of anaerobic fungi from the genus Piromyces is a family 48 glycoside hydrolase
Dna Sequence, 2002Co-Authors: Peter J. M. Steenbakkers, Alex Freelove, Bram Van Cranenbroek, B M C Sweegers, Godfried D. Vogels, Harry Ramanoedj Harhangi, Geoffrey P. Hazlewood, Harry J Gilbert, H Op Den J M CampAbstract:Sequencing of two cDNAs from the anaerobic fungi Piromyces equi and Piromyces sp. strain E2 revealed that they both encode a glycoside hydrolase (GH) family 48 cellulase, containing two C-terminal fungal dockerin domains. N-terminal sequencing of the major component of the Piromyces multi-enzyme cellulase/hemicellulase complex, termed the cellulosome, showed that these 80 kDa proteins corresponded to the GH family 48 enzyme. These data show for the first time that GH family 48 cellulases are not confined to bacteria, and that bacterial and fungal cellulosomes share the same pivotal component.
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Characterization of a cellulosome dockerin domain from the anaerobic fungus Piromyces equi.
Nature structural biology, 2001Co-Authors: S. Raghothama, Tibor Nagy, Geoffrey P. Hazlewood, Harry J Gilbert, Ruth Y. Eberhardt, Peter J. Simpson, Darran Wigelsworth, Peter John White, Michael P WilliamsonAbstract:The recycling of photosynthetically fixed carbon in plant cell walls is a key microbial process. In anaerobes, the degradation is carried out by a high molecular weight multifunctional complex termed the cellulosome. This consists of a number of independent enzyme components, each of which contains a conserved dockerin domain, which functions to bind the enzyme to a cohesin domain within the protein scaffoldin protein. Here we describe the first three-dimensional structure of a fungal dockerin, the N-terminal dockerin of Cel45A from the anaerobic fungus Piromyces equi. The structure contains a novel fold of 42 residues. The ligand binding site consists of residues Trp 35, Tyr 8 and Asp 23, which are conserved in all fungal dockerins. The binding site is on the opposite side of the N- and C-termini of the molecule, implying that tandem dockerin domains, seen in the majority of anaerobic fungal plant cell wall degrading enzymes, could present multiple simultaneous binding sites and, therefore, permit tailoring of binding to catalytic demands.
Anil Kumar Puniya - One of the best experts on this subject based on the ideXlab platform.
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A new anaerobic fungus (Oontomyces anksri gen. nov., sp. nov.) from the digestive tract of the Indian camel (Camelus dromedarius).
Fungal biology, 2015Co-Authors: Sumit Singh Dagar, Sanjay Kumar, Gareth W. Griffith, Joan E. Edwards, Tony M. Callaghan, Rameshwar Singh, Ashok K. Nagpal, Anil Kumar PuniyaAbstract:Two cultures of anaerobic fungi were isolated from the forestomach of an Indian camel (Camelus dromedarius). Phylogenetic analysis using both the internal transcribed spacer (ITS) and large-subunit (LSU) regions of the rRNA locus demonstrated that these isolates were identical and formed a distinct clade within the anaerobic fungi (phylum Neocallimastigomycota). Morphological examination showed that these fungi formed monocentric thalli with filamentous rhizoids and uniflagellate zoospores, broadly similar to members of the genus Piromyces. However, distinctive morphological features were observed, notably the pinching of the cytoplasm in the sporangiophore and the formation of intercalary rhizoidal swellings. Since genetic analyses demonstrated this fungus was only distantly related to Piromyces spp. and closer to the polycentric Anaeromyces clade, we have assigned it to a new genus and species Oontomyces anksri gen. nov., sp. nov. Interrogation of the GenBank database identified several closely related ITS sequences, which were all environmental sequences obtained from camels, raising the possibility that this fungus may be specific to camelids.
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Buwchfawromyces eastonii gen. nov., sp. nov.: a new anaerobic fungus (Neocallimastigomycota) isolated from buffalo faeces
Pensoft Publishers, 2015Co-Authors: Tony Martin Callaghan, S. M. Podmirseg, Sumit Singh Dagar, Anil Kumar Puniya, Daniel Hohlweck, Joan Elizabeth Edwards, Gareth Wyn GriffithAbstract:The novel anaerobic fungus Buwchfawromyces eastonii gen. nov., sp. nov., belonging to order Neocallimastigales (phylum Neocallimastigomycota) is described. Morphologically similar to Piromyces but genetically quite distinct, this fungus (isolate GE09) was first isolated from buffalo faeces in west Wales and then subsequently isolated from sheep, cattle and horse in the same area. Phylogenetic analysis of LSU and ITS sequence confirmed that B. eastonii isolates formed a distinct clade close to the polycentric Anaeromyces spp. The morphology of GE09 is monocentric with monoflagellate zoospores. However, the sporangial stalk (sporangiophore) is often distinctly swollen and the proximal regions of the rhizoidal system twisted in appearance
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effect of administration of anaerobic fungi isolated from cattle and wild blue bull boselaphus tragocamelus on growth rate and fibre utilization in buffalo calves
Archives of Animal Nutrition, 2007Co-Authors: Vimal Kumar Tripathi, Anil Kumar Puniya, J P Sehgal, Kishan SinghAbstract:Abstract Fifteen Murrah buffalo calves (age about 10 months, 163 – 176 kg BW) were divided into three groups. Group I (Control) was fed a complete feed mixture consisted of 50% wheat straw and 50% concentrate mixture (contained per kg: maize 330 g, groundnut cake 210 g, mustard cake 120 g, wheat bran 200 g, de-oiled rice bran 110 g, mineral mixture 20 g and common salt 10 g) along with 2 kg green oats per animal and day to meet the vitamin A requirements. Calves of Groups II and III were fed with the Control diet supplemented with Orpinomyces sp. C-14 and Piromyces sp. WNG-12 cultures, respectively. The digestibility of DM was significantly highest with Piromyces sp. WNG-12 in Group III (62.2%) followed by Orpinomyces sp. C-14 in Group II (60.3%), and Control (53.5%). A similar pattern of increase in digestibility of crude protein and cell-wall contents was observed in treatment groups. The digestible energy in terms of percent total digestible nutrients was also significantly enhanced in Groups II (56.6%...
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in vitro degradation of wheat straw by anaerobic fungi from small ruminants
Archives of Animal Nutrition, 2006Co-Authors: Ankur Thareja, Anil Kumar Puniya, Gunjan Goel, Ravinder Nagpal, J P Sehgal, Pawan K Singh, Kishan SinghAbstract:Abstract Anaerobic ruminal fungi may play an active role in fibre degradation as evidenced by the production of different fibrolytic enzymes in culture filtrate. In the present study, 16 anaerobic fungal strains were isolated from ruminal and faecal samples of sheep and goats. Based on their morphological characteristics they were identified as species of Anaeromyces, Orpinomyces, Piromyces and Neocallimastix. Isolated Neocallimastix sp. from goat rumen showed a maximum activity of CMCase (47.9 mIU ml−1) and filter paper cellulase (48.3 mIU ml−1), while Anaeromyces sp. from sheep rumen showed a maximum xylanolytic activity (48.3 mIU ml−1). The cellobiase activity for all the isolates ranged from 178.0 – 182.7 mIU ml−1. Based on the enzymatic activities, isolated Anaeromyces sp. from sheep rumen and Neocallimastix sp. from goat rumen were selected for their potential of in vitro fibre degradation. The highest in vitro digestibility of NDF (23.2%) and DM (34.4%) was shown for Neocallimastix sp. from goat ru...